Aminoacyl-tRNAs provide the interface between genetic information encoded in the DNA sequence of a gene and the amino acid sequence of the corresponding protein. The vast majority of organisms have been found to lack at least one canonical aminoacyl-tRNA synthetase. The corresponding amino acyl-tRNA is instead synthesized by either a non-canonical aminoacyl-tRNA synthetase or by an alternative pathway. A broad goal of the proposed work is to characterize structurally, biochemically and genetically enzymes and multi-enzyme and tRNA complexes involved in these new pathways for aminoacyl-tRNA synthesis that can then be used to uncover anti-infective targets for human pathogens and for unnatural amino acid incorporation. These general goals will be realized in four specific areas of the proposed work. (i) Selenocyste- inyl-tRNA formation is an essential process in humans, and likely in certain pathogens. Investigations of the enzymes involved, and of an alternative and still unknown route for Sec-tRNA formation will be of fundamental importance for understanding selenocysteine decoding and for exploiting this pathway as a way to combat human pathogens responsible for sleeping sickness and malaria. (ii) Using structure-based high-throughput random mutagenesis and computational design of pyrrolysyl-tRNA synthetase and tRNAPyl, genetic coding systems will be constructed for several lysine derivatives, which will be important for revealing the role of certain post-translational modifications in human and other eukaryotes. Translational quality control by the elongation factor EF-Tu will also be studied along side efforts to design elongation factor mutants that will specifically recognize the desired unnatural aminoacyl-tRNA. (iii) Several amino acids (selenocysteine, cysteine, glutamine, asparagine) are synthesized while attached to the tRNA. Complex formation, between the aminoacyl-tRNA synthetase, tRNA and biosynthetic enzyme(s)-possibly important for substrate channeling and protection of the translation system from mis-acylated tRNAs-will be investigated, to reveal how complex formation affects the enzymatic behavior of the component molecules. (iv) Our discovery and proposed characterization of a novel tRNA ligase(s) will lead to a better understanding of tRNA processing in humans and archaea. PUBLIC HEALTH RELEVANCE: The unexpected diversity of aminoacyl-tRNA synthesis (processes that maintain the coding relation- ship between DNA and protein) opens previously inaccessible frontiers in the biology of translation and post-translational protein modifications, the malfunction of which is linked to several human diseases including cancer, neurodegenerative and metabolic disorders. The proposed projects aim to characterize new routes for aminoacyl-tRNA formation and macromolecular complexes involved in aminoacyl-tRNA synthesis in order to uncover anti-infective targets for human pathogens (including the causative agents of sleeping sickness and malaria) and to design pathways for unnatural amino acid incorporation that will be applied to understanding the complex role of post-translational protein modifications in higher eukaryotes.